2-methyl-4-hexene-and 3-methyl-5-heptene-1,2-diol derivatives

ABSTRACT

2-methyl-4-hexene- and 3-methyl-5-heptene-l,2-diol derivatives are disclosed together with methods for preparing such derivatives. Where the derivative is a 2-methyl-4-nexene- or 3-methyl-5-heptene-l,2-diol conjugated to a label, the conjugates are useful in immunoassays. Where the 2-methyl-4-hexene or 3-methyl-5-heptene-l,2-diol is conjugated to an immunogenic carrier, the conjugates may be employed as an immunogen for use in the preparation of antibodies. The label conjugate and the antibodies can be utilized in an immunoassay for the determination or detection of cyclosporin in a sample suspected of containing cyclosporin.

This is a divisional of pending application Ser. No. 07/093,454, filedSep. 4, 1987, now U.S. Pat. No. 5,089,390, the disclosures of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The body is capable of both specific immune response and non-specificimmune response in dealing with non-self antigens. The body's specificimmune response consists mainly in either producing antibodies orreceptor cells. Non-specific immune response consists of phagocytosis ofbacterial and other particles. Both types of immunity are important inprotecting the body from infecting organisms and in maintaining theintegrity of the body.

There are a number of circumstances where the body's response toinfecting organisms or antigens is deficient. Congenital or acquiredimmune deficiency, failure to distinguish self and non-self antigens,transplantation of immunologically incompatible organs, destruction ofthe hemopoietic system where bone marrow tissue replacement is from anon-identical donor (graph versus host disease) are all examples ofsituations where the body's response is inadequate.

A relatively new drug for treatment of pharmacological immunosuppressionis cyclosporin, particularly, cyclosporin A. Cyclosporin A is anextremely hydrophobic cyclic peptide consisting of 11 amino acids with amolecular weight of approximately 1,200. Cyclosporin A suppresseshumoral as well as cell mediated immunity in all animal species testedto this point, including humans. The drug may be administered bothparenterally or orally. The immunosuppressant effects of cyclosporin Ahave been demonstrated. Cyclosporin A could be potentially useful inseveral types of organ transplantation, various autoimmune diseases, andpossibly rheumatory arthritis.

In administering a cyclosporin, it is frequently necessary to insurethat the blood level of the cyclosporin remains within a certain narrowconcentration range in order to insure effective dosage, while avoidinglevels which may be toxic or produce undesirable effects. Furthermore,it is often necessary to detect potentially toxic levels of cyclosporin.

It is, therefore, desirable to provide a simple and rapid procedure fordetermining or detecting the levels of cyclosporin in serum or otherphysiological fluids. The procedure should provide reproducible valuesand be specific for the cyclosporin which is measured. Thus theprocedure must be capable of distinguishing cyclosporin from otherdrugs, which would otherwise give an erroneous result in an assay forthe detection of a cyclosporin.

2. Brief Description of the Prior Art

A method for the total synthesis of cyclosporins, novel cyclosporins,and novel intermediates and methods for their production are describedin U.S. Pat. Nos. 4,396,542 and 4,554,351. A synthesis ofenantiomerically pure (2S, 3R, 4R, 6E)-3-hydroxy-4-methyl-2-methylamino-6-octenoic acid starting from tartaric acid is disclosed by Wenger inHelvetica Chimica Acta (1983) 66:2308-2321.

SUMMARY OF THE INVENTION

2-methyl-4-hexene- and 3-methyl-5-heptene-l,2-diol (MEHD) derivativesare provided. When the MEHD derivative includes MEHD conjugated to animmunogenic carrier, which is generally a compound of molecular weightgreater than 5,000, the MEHD derivative may be utilized as an immunogento raise antibodies against MEHD. When the MEHD derivative comprisesMEHD conjugated to a label, the MEHD derivatives may be utilized in animmunoassay for the detection of cyclosporin.

The MEHD derivatives of the present invention comprise2-methyl-4-hexene- or 3-methyl-5-heptene-1,2-diol conjugated to a labelor a group of molecular weight greater than 5,000 by means of a linkinggroup of from 0-65 atoms other than hydrogen.

The invention further comprises improvements in assays for thedetermination of cyclosporin in a sample suspected of containingcyclosporin. For example, the assay can comprise the steps of contactinga sample suspected of containing cyclosporin with antibodies forcyclosporin and detecting either directly or indirectly immune complexesof the antibodies and cyclosporin. The improvement of the presentinvention comprises employing the antibodies raised in response to animmunogenic MEHD derivative.

In another example, the assay for the determination of cyclosporin maycomprise the steps of contacting the sample with antibodies forcyclosporin and a conjugate of a label and a compound recognized by theantibodies. The method further comprises detecting either directly orindirectly immune complexes of the label conjugate and the antibodies.The improvement of the present invention comprises employing as thelabel conjugate an MEHD derivative which is MEHD conjugated to a label.

The invention also includes certain improvements in assay reagents foruse in the determination of cyclosporin in a sample suspected ofcontaining cyclosporin. Furthermore, the invention includes kits forconducting an assay for the determination of cyclosporin. The presentinvention also includes methods for preparing MEHD derivatives.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Novel compounds are provided which are derivatives of 2-methyl-4-hexene-and 3-methyl-5-heptene-l,2-diol (MEHD). The MEHO derivatives of thepresent invention in their broadest aspect comprise compounds having2-methyl-4-hexene linked to a label or a group of molecular weightgreater than 5,000. The linking group contains from 0-65 atoms otherthan hydrogen. The compounds of the invention find use in immunoassaysas reagents for use in the detection or determination of cyclosporin ormay be used to raise antibodies that may be utilized as reagents in suchdeterminations.

For the most part, compounds of this invention will have the followingformula: ##STR1## wherein: X is a linking group of from 0-65, preferably0-35, atoms other than hydrogen. When X contains more than 0 atoms, theatoms are independently selected from the group consisting of oxygen,sulfur, phosphorous, nitrogen, silicon, carbon, and halogen, and caninclude such functionalities as, for example, ether, oxocarbonyl,non-oxocarbonyl such as esters, amides, carbamates, sulfone, sulfoxide,phosphate, silicates, and the like, or combinations thereof, preferablyan ether, for example, --CH₂ OA wherein A is a linking group of 0 to 63atoms, preferably 0-33 atoms, other than hydrogen. When A contains morethan 0 atoms, at least 1atom is a carbon atom and A is bonded to --CH₂ Othrough a carbon atom and is bonded to Z. Preferably A contains anon-oxocarbonyl that is bonded to Z, preferably A is C=W wherein W isoxygen, sulfur, or NR wherein R is hydrogen or lower alkyl (C1 to C5),preferably wherein C=W is bonded to NRX', wherein X' is a linking grouphaving from 0 to 60 atoms other than hydrogen, preferably 0 to 30 atomsother than hydrogen. When X' contains more than 0 atoms the atoms areselected from the group consisting of oxygen, sulfur, phosphorus,nitrogen, silicon, and carbon. The term non-oxocarbonyl intends acarbonyl group substituted with at least one heteroatom; for thepurposes of this disclosure non-oxocarbonyl shall include but not belimited to ##STR2## and the iminocarbonyl and thiocarbonyl analogsthereof. Z is a luminescent label or a group of molecular weight greaterthan 5,000, more preferably greater than 10,000, preferably, theluminescent label is selected from the group consisting of fluorogenicsubstrates, fluorescers, chemiluminescers, and fluorescent particles andthe group of molecular weight greater than 5,000 is selected from thegroup consisting of poly(amino acids) such as enzymes and immunogeniccarriers selected from the group consisting of immunogenic poly(aminoacids), lipopolysaccharides and particles, or Z can be a ligand forwhich a receptor is available, such as biotin or a hapten, such as anorganic molecule of less than 1000 molecular weight.

Y is oxygen or sulfur, preferably oxygen; and

n is a number from 1 up to the molecular weight of Z divided by 1,000,preferably divided by 4,000, preferably 1 when Z is a luminescent labeland 2 to 20, usually 3 to 10 when Z is a poly(amino acid).

The above compounds include both optically active and optically inactiveisomers. The optically active isomers where X is --CH₂ OA-- include (2S,2S) or (2S, 2R) or (2R, 2S) or (2R, 2R), and also include the cis- andtrans- isomers.

A particular embodiment of the present invention includes compounds ofthe formula, which are derivatives of 3-methyl-5-neptene-1,2-diols:##STR3## wherein: X₁ is a linking group of from 0-35 atoms other thanhydrogen. When X₁ contains more than 0 atoms, at least 1 atom is acarbon atom and the remaining atoms are each independently selected fromthe group consisting of carbon, oxygen, nitrogen, and sulfur, whereinthe linking group is bound to 0 through a carbon atom and is bonded toZ, and wherein the linking group can include a functionality such asether, nonoxocarbonyl, preferably amide, including the nitrogen andsulfur analogs, preferably a linking group of from 2 to 10 atoms otherthan hydrogen wherein the group includes an amide functionalityincluding carbamate.

Z₁ is a poly(amino acid) of molecular weight greater than 5,000, whichis immunogenic or an enzyme; and

n₁ is a number from 1 up to the molecular weight of Z₁ divided by 1,000,preferably divided by 4,000, preferably 2 to 20, more preferably 3 to10.

Another particular embodiment of the present invention includescompounds of the formula: ##STR4## wherein: W₁ is O, S, or NH,preferably O;

m₁ is 0 to 10, preferably 1 to 5;

Z₂ is a polyamino acid, which is an immunogenic or an enzyme; or biotinor a hapten;

n₂ is a number from 1 up to the molecular weight of Z₂ divided by 1,000,preferably divided by 4,000.

The group having a molecular weight greater than 5,000 includespolyamino acids, lipopolysaccharides, and particles. Such a group can bea label or an immunogenic carrier.

The poly(amino acids) will generally range from about 5,000 molecularweight, having no upper molecular weight limit, normally being less than10,000,000, usually not more than about 600,000 daltons. There willusually be different ranges, depending on whether an immunogen or anenzyme is involved, with immunogens ranging from about 5,000 to 10⁷,usually from about 20,000 to 600,000, and more usually from about 25,000to 250,000 molecular weight; while enzymes will generally range fromabout 10,000 to 600,000, more usually from about 10,000 to 300,000molecular weight. There will usually be at least about one MEHDconjugate group per 150,000 molecular weight, more usually at least oneconjugate group per 50,000 molecular weight. With intermediate molecularweight immunogens (35,000 to 1,000,000), the number of conjugate groupswill generally be from about 1 to 200, more usually from 2 to 100. Withlower molecular weight immungens below 35,000, the number of conjugategroups will generally be in the range of from about 1 to 10, usually inthe range of 1 to 5.

Various protein types may be employed as the poly(amino acid)immunogenic material. These types include albumins, serum proteins,e.g., globulins, ocular-lens proteins, lipoproteins, etc. Illustrativeproteins include bovine serum albumin, keyhole limpet hemocyanin, eggovalbumin, bovine γ-globulin, etc. Alternatively, synthetic poly(aminoacids) may be utilized.

The immunogenic carrier can also be a polysaccharide, which is a highmolecular weight polymer built up by repeated condensations ofmonosaccharides. Examples of polysaccharides are starches, glycogen,cellulose, carbohydrate gums, such as gum arabic, agar, and so forth.The polysaccharide can also contain polyamino acid residues and/or lipidresidues.

The immunogenic carrier can also be a nucleic acid either alone orconjugated to one of the above mentioned poly(amino acids) orpolysaccharides.

The immunogenic carrier can also be a particle. The particles aregenerally at least about 0.02 microns and not more than about 100microns, usually at least about 0.05 microns and less than about 20microns, preferably from about 0.3 to 10 microns diameter. The particlemay be organic or inorganic, swellable or non-swellable, porous ornon-porous, preferably of a density approximating water, generally fromabout 0.7 to about 1.5 g/ml, and composed of material that can betransparent, partially transparent, or opaque. The particles can bebiologic materials such as cells and microorganisms, e.g., erythrocytes,leukocytes, lymphocytes, hybridomas, streptococcus, staphylococcusaureus, E. coli, viruses, and the like. The particles can also beparticles comprised of organic and inorganic polymers, liposomes, latexparticles, phospholipid vesicles, chylomicrons, lipoproteins, and thelike.

The polymers can be either addition or condensation polymers. Particlesderived therefrom will be readily dispersible in an aqueous medium andmay be adsorptive or functionalizable so as to bind to MEHD, eitherdirectly or indirectly through a linking group.

The particles can be derived from naturally occurring materials,naturally occurring materials which are synthetically modified andsynthetic materials. Among organic polymers of particular interest arepolysaccharides, particularly cross-linked polysaccharides, such aagarose, which is available as Sepharose, dextran, available as Sephadexand Sephacryl, cellulose, starch, and the like; addition polymers, suchas polystyrene, polyvinyl alcohol, homopolymers and copolymers ofderivatives of acrylate and methacrylate, particularly esters and amideshaving free hydroxyl functionalities, and the like.

The particles will usually be Polyfunctional and will be bound to or becapable of binding to MEHD. A wide variety of functional groups areavailable or can be incorporated. Functional groups include carboxylicacids, aldehydes, amino groups, cyano groups, ethylene groups, hydroxylgroups, mercapto groups and the like. The manner of linking a widevariety of compounds to particles is well known and is amply illustratedin the literature. See for example Cautrecasas, J. Biol. Chem. (1970)245:3059.

The poly(amino acid) can be an enzyme that is part of a signal producingsystem. The function of the signal producing system is to produce aproduct which provides a detectable signal related to the amount ofbound and unbound label. Where enzymes are employed, the involvedreactions will be, for the most part, hydrolysis or redox reactions.

Coupled catalysts can also involve an enzyme with a non-enzymaticcatalyst. The enzyme can produce a reactant which undergoes a reactioncatalyzed by the non-enzymatic catalyst or the non-enzymatic catalystmay produce a substrate (includes coenzymes) for the enzyme. A widevariety of non-enzymatic catalysts which may be employed are found inU.S. Pat. No. 4,160,645, issued Jul. 10, 1979, the appropriate portionsof which are incorporated herein by reference.

The enzyme or coenzyme employed provides the desired amplification byproducing a product, which absorbs light, e.g., a dye, or emits lightupon irradiation, e.g., a fluorescer. Alternatively, the catalyticreaction can lead to direct light emission, e.g., chemiluminescence. Alarge number of enzymes and coenzymes for providing such products areindicated in U.S. Pat. No. 4,275,149 bridging columns 19 to 23, and U.S.Pat. No. 4,318,980 columns 10 to 14, which disclosures are incorporatedherein my reference.

A number of enzyme combinations are set forth in U.S. Pat. No.4,275,149, bridging columns 23to 28, which combinations can find use inthe subject invention. This disclosure is incorporated herein byreference.

Of particular interest are enzymes which involve the production ofhydrogen peroxide and the use of the hydrogen peroxide to oxidize a dyeprecursor to a dye. Particular combinations include saccharide oxidases,e.g., glucose and galactose oxidase, or heterocyclic oxidases, such asuricase and xanthine oxidase, coupled with an enzyme which employs thehydrogen peroxide to oxidize a dye precursor, that is, a peroxidase suchas horse radish peroxidase, lactoperoxidase, or microperoxidase.Additional enzyme combinations may be found in the subject matterincorporated by reference.

When a single enzyme is used as a label, such enzymes that may find useare hydrolases, transferases, lyases, isomerases, ligases or synthetasesand oxidoreductases, preferably hydrolases. Alternatively, luciferasesmay be used such as firefly luciferase and bacterial luciferase.Primarily, the enzymes of choice, based on the I.U.B. classificationare: Class 1. Oxidoreductases and Class 3. Hydrolases; particularly inClass 1, the enzymes of interest are dehydrogenases of Class 1.1, moreparticularly 1.1.1, 1.1.3, and 1.1.99 and peroxidases, in Class 1.11. Ofthe hydrolases, particularly Class 3.1, more particularly 3.1.3 andClass 3.2, more particularly 3.2.1.

Illustrative dehydrogenases include malate dehydrogenase,glucose-6-phosphate dehydrogenase, and lactate dehydrogenase. Of theoxidases, glucose oxidase is exemplary. Of the peroxidases, horse radishperoxidase is illustrative. Of the hydrolases, alkaline phosphatase,β-glucosidase and lysozyme are illustrative.

Those enzymes which employ nicotinamide adenine dinucleotide (NAD) orits phosphate (NADP) as a cofactor, particularly the former can be used.One preferred enzyme is glucose-6-phosphate dehydrogenase.

A label may be any molecule conjugated to an analyte or an antibody, orto another molecule. In the subject invention, the label can be a memberof the signal producing system that includes a signal producing means.The label may De isotopic or nonisotopic, preferably nonisotopic. By wayof example and not limitation, the label can be a catalyst such as anenzyme, a co-enzyme, a chromogen such as a fluorescer, dye orchemiluminescer, a dispersible particle that can be non-magnetic ormagnetic, a solid support, a liposome, a ligand, a hapten, and so forth.

The signal producing system may have one or more components, at leastone component being a label. The signal producing system includes all ofthe reagents required to produce a measurable signal including signalproducing means capable of interacting with the label to produce asignal.

The signal producing system provides a signal detectable by externalmeans, normally by measurement of electromagnetic radiation, desirablyby visual examination. For the most part, the signal producing systemincludes a chromophoric substrate and enzyme, where chromophoricsubstrates are enzymatically converted to dyes which absorb light in theultraviolet or visible region, phosphors or fluorescers.

The signal producing means is capable of interacting with the label toproduce a detectable signal. Such means include, for example,electromagnetic radiation, heat, chemical reagents, and the like. Wherechemical reagents are employed, some of the chemical reagents can beincluded as part of a developer solution. The chemical reagents caninclude substrates, coenzymes, enhancers, second enzymes, activators,cofactors, inhibitors, scavengers, metal ions, specific bindingsubstances required for binding of signal generating substances, and thelike. Some of the chemical reagents such as coenzymes, substances thatreact with enzymic products, other enzymes and catalysts, and the likecan be bound to other molecules or to a support.

The signal producing system including the label can include one or moreparticles, which are insoluble particles of at least about 50 nm and notmore than about 50 microns, usually at least about 100 nm and less thanabout 25 microns, preferably from about 0.2 to 5 microns, diameter. Theparticle may be organic or inorganic, porous or non-porous, preferablyof a density approximating water, generally from about 0.7 to about 1.5g/ml, and composed of material that can be transparent, partiallytransparent, or opaque. Generally, particles utilized as a label willhave similar characteristics to those described above for an immunogeniccarrier.

Many different types of particles may be employed for modulating lightemission. Of particular interest are carbon particles, such as charcoal,lamp black, graphite, colloidal carbon and the like. Besides carbonparticles metal sols may also find use, particularly of the noblemetals, gold, silver, and platinum. Other metal-derived particles mayinclude metal sulfides, such as lead, silver or copper sulfides or metaloxides, such as iron or copper oxide.

Fluoresceinated latex particles are taught in U.S. Pat. No. 3,853,987and are available commercially as Coraspheres from Covalent TechnologyCorp.

The label can also be fluorescent either directly or by virtue offluorescent compounds or fluorescers bound to a particle in conventionalways. The fluorescers will usually be capable of, or functionalized torender them capable of, being bound to MEHD or to particle.

The fluorescers of interest will generally emit light at a wavelengthabove 350 nm, usually above 400nm and preferably above 450 nm.Desirably, the fluorescers have a high quantum efficiency, a largeStokes shift and are chemically stable under the conditions of theirconjugation and use. The term luminescent label is intended to includesubstances that emit light upon activation by electromagnetic radiationor chemical activation and includes fluorescent and phosphorescentsubstances, scintillators, and chemiluminescent substances.

Fluorescers of interest fall into a variety of categories having certainprimary functionalities. These primary functionalities include 1- and2-aminonaphthalene, p,p-diaminostilbenes, pyrenes, quaternaryphenanthridine salts, 9-aminoacridines, p,p'-diaminostilbenes imines,anthracenes, oxacarboxyanine, merocyanine, 3-aminoequilenin, perylene,bis-benzoxaole, bis-p-oxazolyl benzene, 1,2-benzophenazine, retinol,bis-3-aminopyridinium salts, hellebrigenin, tetracycline, sterophenol,benzimidazolylphenylamine, 2-oxo-3-chromen, indole, xanthene,7-hydroxycoumarin, 4,5-benzimidazoles, phenoxazine, salicylate,strophanthidin, porphyrins, triarylmethanes, flavin and rare earthchelates oxides and salts. Exemplary fluorescers are enumerated in U.S.Pat. No. 4,318,707, columns 7 and 8, the disclosure of which isincorporated herein by reference.

Energy absorbers or quenchers can be employed either separately or inconjunction with one another. The absorber or quencher can additionallybe bound to a solid insoluble particle of at least about 50nm indiameter. When the distance between the absorber and the quencherresulting from specific binding events (such as antibody-antigenbinding), the fluorescence of the absorber is quenched by the quencher.The quencher may be the same or different, usually different, from thefluorescer.

An alternative source of light as a detectable signal is achemiluminescent source, and, therefore, a label can be achemiluminescent compound. The chemiluminescent source involves acompound which becomes electronically excited by a chemical reaction andmay then emit light which serves as the detectable signal or donatesenergy to a fluorescent acceptor.

A diverse number of families of compounds have been found to providechemiluminescence under a variety of conditions. One family of compoundsis 2,3-dihydro-l,4-phthalazinedione. The most popular compound isluminol, which is the 5-amino analog of the above compound. Othermembers of the family include the 5-amino-6,7,8-trimethoxy- and thedimethylamine-[ca]benzo analog. These compounds can be made to luminescewith alkaline hydrogen peroxide or calcium hypochlorite and base.Another family of compounds is the 2,4,5-triphenylimidazoles, withlophine as the common name for the parent product. Chemiluminescentanalogs include para-dimethylamino- and para-methoxy-substituents.Chemiluminescence may also be obtained with oxalates, usually oxalyl,active esters, e.g., p-nitrophenyl and a peroxide, e.g., hydrogenperoxide, under basic conditions. Alternatively, luciferins may be usedin conjunction with luciferase or lucigenins.

An example of the preparation of a compound of the invention wherein X(Formula I) includes a --CH₂ O-- group is outlined in the followingschematic: ##STR5## wherein: a) ∝ is halogen, preferably bromine.Compound 0 is reacted under suitable anhydrous conditions with anappropriate organometallic reagent to prepare Compound 1. In the exampleshown, the organometallic reagent is BrMgOCH₂ C.tbd.CMgBr. However,other organometallic reagents can be utilized.

b) Compound 1 is treated under reducing conditions and reagents, such aslithium aluminum hydride in an anhydrous medium to reduce the triplebond and give Compound 2.

c) Compound 2 is treated to form the epoxide of Compound 3. Suchtreatment includes contact with, for example, a hydroperoxide such as analkyl hydroperoxide, e.g., tert-butyl hydroperoxide.

d) Compound 3 is derivatized to protect the terminal hydroxyfunctionality. Accordingly, β is a removable protecting group such as asubstituted silyl group, e.g., diphenyl t-butylsilyl. Compound 3 istreated under appropriate silylating conditions with a substituted silylhalide, e.g., diphenyl t-butylsilyl chloride, to produce compound 4.

e) Compound 4 is treated under conditions for methylating the epoxidefunctionality. Accordingly, 4 can be reacted under appropriateconditions with, e.g., (CH₃)₂ CuCNLi₂ to give Compound 5.

f) The free hydroxy group of Compound 5 is protected with a removableprotecting group γ such as, for example, alkoxyalkyl group (C2 to C8).Accordingly, 5 is reacted with, e.g., CH₂ ═CH--OC₂ H₅, or homologthereof, under appropriate conditions to give Compound 6.

g) Protecting group β of Compound 6 is removed under appropriateconditions to give Compound 7.

h) The free hydroxy group of Compound 7 is treated with an activatedformate derivative ##STR6## wherein E and G are leaving groups such asaryloxy, halo, etc., to provide an activated formate ester capable offorming an amide by reaction with an amino group; such activated formateester groups include, e.g., N-oxy-succinimide, p-nitropnenoxy, and thelike. Thus, 7 is reacted under suitable condition with, for example,##STR7## wherein E is halogen and G is p-nitrophenoxyl to give Compound8. i) Compound 8 is reacted under appropriate conditions with a compoundcontaining an amino group such as, for example, H₂ N(CH₂)_(p) CO₂ H,wherein p is 1 to 20, to give Compound 9.

j) The hydroxy protecting group of Compound 9 is removed underappropriate conditions such as treatment with an acidic medium, e.g.,dilute mineral acid.

Compound 10 can then be utilized to prepare the derivatives of thepresent invention by reaction under suitable conditions. For example,enzyme conjugates of Compound 10 can be prepared by forming an activatedester of the carboxyl group of Compound 10 and reacting Compound 10 withthe appropriate enzyme to yield an amide enzyme derivative of Compound10. Other derivatives of Compound 10 can be prepared in a manner similarto the enzyme derivatives. In such a way one can prepare derivatives ofCompound 10 having a luminescent label, ligand label, immunogenicpoly(amino acid), polysaccharide, particle, and the like. Variations inthe above scheme can be made by those skilled in the art to producecompounds falling within the scope of the present invention.

Another aspect of the present invention includes antibodies prepared inresponse to Compound 1 wherein Z is an immunogenic carrier. Furthermore,the present invention includes conjugates of such antibodies and alabel.

An antibody is an immunoglobulin which specifically binds to and isthereby defined as complementary with a particular spatial and polarorganization of another molecule. The antibody can be monoclonal orpolyclonal and can be prepared by techniques that are well known in theart such as immunization of a host and collection of sera from which theimmunoglobulin can be separated by known techniques (polyclonal) or bypreparing continuous hybrid cell lines and collecting the secretedprotein (monoclonal). Antibodies may include a complete immunoglobulinor fragment thereof, which immunoglobulins include the various classesand isotypes, such as IgA, IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgM,etc. Fragments thereof may include Fab, Fv and F(ab')₂, Fab', and thelike.

Monoclonal antibodies can be obtained by the process discussed byMilstein and Kohler and reported in Nature, 256:495-497, 1975. Thedetails of this process are well known and will not be repeated here.However, basically it involves injecting a host, usually a mouse orother suitable animal, with an immunogen. The mouse is subsequentlysacrificed and cells taken from its spleen. Alternatively, the host maybe unsensitized spleen cells, which are sensitized to the immunogen invitro. The resulting cells are fused with myeloma cells. The result is ahybrid cell, referred to as a "hybridoma" that can be cultured in vitro.The population of hybridomas is screened and manipulated so as toisolate individual clones each of which secretes a single antibody tothe antigen.

The antibodies of the present invention recognize the cyclosporinsincluding cyclosporin A and derivatives and metabolites of cyclosporins.Cyclosporin A has the following structure: ##STR8##

The antibodies of the present invention are capable of specificallyrecognizing cyclosporin and closely related compounds containing theunmodified C₉ amino acid.

Conjugates of the antibodies of the present invention and a label can beprepared by methods already descended above for the conjugation ofpoly(amino acids) to HEHD.

The antibodies or the present invention can be utilized in thedetermination of cyclosporin in a sample suspected of containingcyclosporin. The assay can comprise the steps of contacting the samplewith antibodies for cyclospoin and detecting either directly orindirectly immune complexes of the antibodies and cyclosporin. Theimprovement provided in the present invention is the utilization of thepresent antibodies as the antibodies for cyclosporin. The immunecomplexes are detected directly, for example, where the antibodiesemployed are conjugated to a label. The immune complex is detectedindirectly by examining for the effect of immune complex formation in anassay medium, on a signal producing system or by employing a labeledantibody that specifically binds to an antibody of the invention.

In another configuration of an assay for the determination ofcyclosporin in a sample suspected of containing cyclosporin, the sampleis contacted with antibodies for cyclosporin and a conjugate of thisinvention recognized by the antibodies. The method further includesdetecting either directly or indirectly immune complexes of theconjugate and the antibodies. The improvement provided in the presentinvention is employing as the label conjugate Compound 1 wherein Z is alabel.

The present assay invention has application to all immunoassays forcyclosporin. The assay can be performed either without separation(homogeneous) or with separation (heterogeneous) of any of the assaycomponents or products. Exemplary of heterogeneous assays are enzymelinked immunoassays such as the enzyme linked immunosorbent assay(ELISA), see "Enzyme-Immunoassay" by Edward T. Maggio, CRC PressIncorporated, Boca Raton, Fla., 1980. Homogeneous immunoassays areexemplified by enzyme multiplied immunoassay techniques (e.g. see U.S.Pat. No. 3,817,817), immunofluorescence methods such as those disclosedin U.S. Pat. No. 3,993,345, enzyme channeling techniques such as thosedisclosed in U.S. Pat. No. 4,233,402, and other enzyme immunoassays asdiscussed in Maggio, supra.

The assay for the analyte will normally be carried out in an aqueousbuffered medium at a moderate pH, generally that which provides optimumassay sensitivity.

The aqueous medium may be solely water or may include from 0 to 40volume percent of a cosolvent. The pH for the medium will usually be inthe range of about 4 to 11, more usually in the range of about 5 to 10,and preferably in the range of about 6.5 to 9.5. The pH will usually bea compromise between optimum binding of the binding members and the pHoptimum for other reagents of the assay such as members of the signalproducing system.

Various buffers may be used to achieve the desired pH and maintain thepH during the determination. Illustrative buffers include borate,phosphate, carbonate, tris, barbital and the like. The particular bufferemployed is not critical to this invention, but in an individual assayone or another buffer may be preferred.

Moderate temperatures are normally employed for carrying out the assayand usually constant temperatures during the period of the measurement,particularly for rate determinations. Incubation temperatures willnormally range from about 5° to 45° C., more usually from about 15° to40° C. Temperatures during measurements will generally range from about10° to 50° more usually from about 15° to 40° C.

The concentration of cyclosporin which may be assayed will generallyvary from about 10⁻⁵ to 10⁻¹³ M, more usually from about 10⁻⁷ to 10⁻¹¹M. Considerations, such as whether the assay is qualitative,semiquantitative or quantitative, the particular detection technique andthe concentration of the cyclosporin will normally determine theconcentrations of the various reagents.

While the concentrations of the various reagents in the assay mediumwill generally be determined by the concentration range of interest ofcyclosporin, the final concentration of each of the reagents willnormally be determined empirically to optimize the sensitivity of theassay over the range. That is, a variation in concentration ofcyclosporin which is of significance should provide an accuratelymeasurable signal difference.

While the order of addition may be varied widely, there will De certainpreferences depending on the nature of the assay. The simplest order ofaddition is to add all the materials simultaneously and determine theeffect that the assay medium has on the signal as in a homogeneousassay. Alternatively, the reagents can be combined sequentially.Optionally, an incubation-step may be involved subsequent to eachaddition, generally ranging from about 30 seconds to 6 hours, moreusually from about 2 minutes to 1 hour.

In a homogeneous assay after all of the reagents have been combinedeither simultaneously or sequentially, the effect of the assay medium onthe signal is determined. The effect of the assay medium on the signalis related to the amount of cyclosporin in the sample tested. Forexample, in the enzyme multiplied immunoassay technique for thedetection of cyclosporin, a sample suspected of containing cyclosporinis combined either simultaneously or sequentially with an antibody ofthe present invention and a conjugate of an enzyme and an MEHDderivative such as compound I wherein Z is an enzyme. A particularlypreferred enzyme is glucose-6-phosphate dehydrogenase. The cyclosporinin the sample and the MEHD-enzyme conjugate compete for sites on theantibody. The difference in enzyme activity resulting from the presenceor absence of cyclosporin in the sample is then determined By knowntechniques and is related to the amount of cyclosporin in the sample.

Heterogeneous assays usually involve one or more separation steps. Theheterogeneous assay can be competitive or non-competitive. In thecompetitive assay an antibody of the invention can be bound to asupport, which is then contacted with a medium containing sample and aconjugate of MEHD and an enzyme such as-compound I wherein Z is anenzyme label. Cyclosporin in the sample competes with the conjugate forthe sites on the antibody bound to the support. After separation of thesupport and the medium, the enzyme activity of the support or the mediumcan be determined by conventional techniques and is related to theamount of cyclosporin in the sample.

In another competitive heterogeneous approach, an antibody of theinvention is bound to a support. A medium is prepared containing asample suspected of containing cyclosporin and a conjugate of MEHD and asmall organic molecule (molecular weight less than 1500) such as biotin(compound I wherein Z is biotin). Cyclosporin and the conjugate competefor the antibody sites. After a period of time, the support is separatedfrom the medium, washed, and contacted with a second medium containing areceptor or binding partner for the small organic molecule bound to alabel such as an enzyme. If the small organic molecule is biotin, thesupport can be contacted with avidin bound to an enzyme. The support isseparated from the second medium and enzyme activity of either thesupport or the second medium is determined by conventional methods. Theenzyme activity is related to the amount of cyclosporin in the sample.

An example of a non-competitive approach is a sandwich assay involvingtwo antibodies, one of which can be labeled and further one of which canbe bound to a support or caused to become bound to a support.

In another embodiment, agglutination can be utilized to determine thepresence or amount of cyclosporin in a sample. Antibodies raised inresponse to compound I wherein Z is an immunogenic carrier can beconjugated to particles. Another particle conjugate that can be employedis compound I where Z is a particle. In one approach, a sample suspectedof containing cyclosporin can be combined with an antibody-particleconjugate mentioned above. After an appropriate incubation, the extentof agglutination of the particles due to the presence of cyclosporin canbe determined directly. On the other hand, the particles can beseparated from the medium, washed, and combined with a conjugate of aparticle and MEHD. Agglutination can then be determined as a measure ofthe amount of cyclosporin in the sample. The above descriptionexemplifies only two of many agglutination protocols that may be carriedout utilizing the compounds of the invention.

One aspect of the present invention includes compounds of the formulawhich can be utilized as intermediates in the preparation of the MEHDderivatives of the invention: ##STR9## wherein: Q is a bond or loweralkyl (C1 to C5);

Q' is OH or OR₁ wherein R₁ is lower alkyl, alkoxyalkyl (C2 to C8) suchas ethoxyethyl, when Q is a bond Q' can be taken together with Q and anoxygen atom to form a three-membered ring;

T is a removable protecting group such as silicon having substituentseach independently selected from the group consisting of aryl (6-20atoms) such as phenyl, alkaryl (7-25 atoms) such as benzyl, and loweralkyl such as t-butyl, or W₂ CY₂₀ R₂ wherein W₂ is O, S, or NH, Y₂ is Oor S, and R₂ is phenyl, p-nitrophenyl, or HN(CH₂)_(q) CO₂ H wherein q is1 to 20 such as . H₂ NCH₂ CO₂ H; or T can be a group removable by metalreduction, such as benzyl, naphthylmethyl, and the like; and

T' is hydrogen or, when T is lower alkyl or aryl, T' can be OT, whereinthe T's can be linked together to form a five or six-membered ring.

To enhance the versatility of the subject invention, the reagents can beprovided in packaged combination, in the same or separate containers, sothat the ratio of the reagents provides for substantial optimization ofthe method and assay. The kit comprises as one reagent an antibodyraised in response to Compound I wherein Z is an immunogenic carrier.This antibody can be labeled or unlabeled. The kit may also includeCompound I wherein Z is a label. The kit can further include otherseparately packaged reagents for conducting an assay including membersof the signal producing system, other antibodies either labeled orunlabeled, supports, ancillary reagents, and so forth.

A support is a porous or non-porous water insoluble material. Thesupport can be hydrophilic or capable of being rendered hydrophilic andincludes inorganic powders such as silica, magnesium sulfate, andalumina; natural polymeric materials, particularly cellulosic materialsand materials derived from cellulose, such as fiber containing papers,e.g., filter paper, chromatographic paper, etc.; synthetic or modifiednaturally occurring polymers, such as nitrocellulose, cellulose acetate,poly (vinyl chloride), polyacrylamide, cross linked dextran, agarose,polyacrylate, polyethylene, polypropylene, poly(4-methylbutene),polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon,poly(vinyl butyrate), etc.; either used by themselves or in conjunctionwith other materials; glass, ceramics, metals, and the like.

Various ancillary materials will frequently be employed in an assay inaccordance with the present invention. For example, buffers willnormally be present in the assay medium, as well as stabilizers for theassay medium and the assay components. Frequently, in addition to theseadditives, additional proteins may be included, such as albumins, orsurfactants, particularly non-ionic surfactants, binding enhancers,e.g., polyalkylene glycols, or the like.

EXAMPLE 1

Preparation of (E)-Hept-5-ene-2,yn-1-ol

Trans crotyl bromide (59.4g) was reacted with a solution ofprop-2-yn-1-ol (22.4g) and two equivalents (eq.) of ethylmagnesiumbromide to give mostly (E)-hept-5-ene-2-yn-1-ol (1) as a colorless oil(40g, 80%), b.p. 71°-71.5° C. (20 mm Hg). NMR (CDCl₃, δ): 1.6 (dd, CH₃),2.4 (t, OH), 2.9 (m, 2H), 4.2 (m,2H), 5.50 (m, 2H). I.R. (neat) 3350s,3050m, 2940s, 2900s, 2300w, 2250w, 1650w, cm-¹.

EXAMPLE 2

Preparation of (E,E)-hepta-2,5.-diene-1-ol

Compound 1 (22.0 g) was reacted with lithium aluminum hydride (23.0 g, 3eq), in ether to give (E,E)-hepta-2,5-diene-1-ol (2) as a colorless oil(21 g) b.p. 98°-100° C. (35 mm Hg). NMR (CDCl₃, δ): 1.6 (dd, 3H), 1.9(b, OH), 2.7 (m, 2H), 4.1 (m, 2H), 5.4 (m, 2H), 5.7 (m, 2H). IR (neat)3350s, 3025m, 2980m, 2950s, 2925s, 2900s, 2850s, 1670w, 1430m, cm-¹.

EXAMPLE 3

Preparation of Hept-2S,3S-epoxy-5(E)-ene-1-ol

Compound 2 (14.5 g) was epoxidized using 5% titanium (IV) isopropoxide(1.98 g) and t-butylhydroperoxide and 5% L(+) diethyl tartarate (2.10 g)and 4A° molecular sieves to give hept-2S,3S-epoxy-5(E)-ene-1-ol (3) as alow melting solid 14.4 g, m.p. 20°-25° C. NMR (CDCl₃, δ): 1.6 (m, 3H),2.3 (m, 2H), 2.9 (m, 2H), 3.3-4.1 (m, 2H, OH), 5.5 (m, 2H). IR (neat)3400s, 3050m, 2975s, 2925s, 2850s, cm-¹, [∝]_(D) ²⁵ =-27 (C=1, CHCl₃).

EXAMPLE 4

Preparation of the t-Butyl Diphenylsilyl Ether of Hept-2S ,3S,-epoxy-5(E)-ene-1-ol

Compound 3 (14.2 g) was reacted with t-butyl diphenyl chlorosilane (33.8g) and imidazol (16.73 g) in DMF to give the silane derivative 4 (β=(C₆H₅)₂ SiC(CH₃)₃) (40 g) as a colorless liquid. NMR (CDCl₃, δ): 1.1(s,9H), 1.7 (m, 3H), 2.3 (m, 2H), 2.7-3.1 (m, 2H), 3.8 (d, 2H), 5.4-5.8 (m,2H), 7.3-7.9 (m, 10H). IR (neat); 3100m, 3075m, 3000m, 2970s, 2940s,2900w, 2850s, 1590w, 1470w, 1460m, 1430s, cm-¹, [∝]_(D) ²⁵ =-6.4 (C=1,CHCl₃).

EXAMPLE 5

Preparation of 2R, 3R, 5E-2-ol-3-methyl-5-heptene-1-t-butyl diphenylsilyl ether

Compound 4 (14.62 g) was reacted with lithium dimethyl cyano cuprate(prepared from 8.0 g of CaCN) (2eq) in ether:THF mixture to givecompound 5 (β=(C₆ H₅)₂ SiC(CH₃)₃) as a colorless oil which was purifiedon silica gel; ether:hexane (2:98) was used as eluting solvent. NMR(CDCl₃,δ): 0.8(d, 3H) 1.1 (s, 9H), 1.6(d, 3H), 1.8∝2.4 (m. 2H), 2.6 (d,--OH), 3.4-3.8 (m, 3H), 5.4 (m, 2H), 7.3-7.8 (m, 10H). IR (neat): 3575w,3450w, 3075w, 3050w, 3010w, 3000w, 2970s, 294Os, 2900m, 2860s, 1590w,1470m, 1460m, 1406s, cm-¹, [α]_(D) ²⁵ =-8.1 (C=1,CHCl₃).

EXAMPLE 6

Preparation of 2R,3R,5E-2-(1-ethoxymethoxy)-3-methyl-5-heptene-1-t-butyl diphenyl silylether

Compound 5 (β=(C₆ H₅)₂ SiC(CH₃)₃) (50 g) was reacted with ethyl vinylether (11.5 g) and pyrdinium p-toluene sulfonate (0.260 mg) to givecompound 6 (γ=-CH(CH₃)OC₂ H₅ (β=(C₆ H₅)₂ SiC(CH₃)₃)) as a colorless oilwhich was a mixture of 1:1 diastereomers (5.0 g). NMR(CDCl₃,δ): 0.8 (dd,3H), 1.2 (dq, 3H), 1.3 (d, 3H), 1.6 (d, 3H), 1.7-2.2 (m, 2H), 3.3-3.8(m, 5H), 4.6 (q, 1/2H), 4.8 (q, 1/2H), 5.4 (m, 2H), 7.3-7.8 (m, 10H). IR(neat): 3075 m, 3050m, 3010m, 2960s, 2940s, 2900s, 2850s, 1590w, 1470m,1460m, 1430s, cm-¹ . [α]_(D) ²⁵ =-0.71 (C=1, CHCl₃).

EXAMPLE 7

Preparation of 2R,3R,5E-2-(1'-ethoxymethoxy)-3-methyl-5-heptene-1-ol

Compound 6 (γ=--CH(CH₃)OC₂ H₅) (5.0 g) was reacted with tetrabutylammonium fluoride 23 ml (1M in THF, 2 eq.) in THF to give Compound 7(γ=CH(CH₃)OC₂ H₅) as a colorless oil 2.3 g and a mixture of 1:1diastereomers. Physical data and IR, NMR spectra are identical with thereported data in the literature, Roger Wenger, Helvetica Chemica Acta,2308 (1983).

EXAMPLE 8

Preparation of 2R, 3R,5E-2(1'-ethoxymethoxy)-3-methyl-5-hepten-1-p-nitrophenyl formate

Compound 7 (γ=--CH(CH₃)OC₂ H₅) (216 mg) was reacted withp-nitrophenylchloroformate (230 mg) (1.1 eq.) in ether and triethylamine (1.2 eq) at 0° to give the carbamate 8 (γ=--CH(CH₃)OC₂ H₅, G=OC₆H₄ NO₂) which after evaporation of the solvent was used without furtherpurification.

Example 9

Preparation of 2R,3R, 5E-2-(1'-ethoxymethoxy)-3-methyl-5-hepten-1-glycyl carbamate

Carbamate 8 (γ=--CH(CH₃)OC₂ H₅, G=OC₆ H₄ NO₂) was dissolved in DMF andadded to a solution of glycine in water at pH=8.5 at room temperaturefor 24 hours to give Compound 9 (γ=--CH(CH₃)OC₂ H₅, p=1) (150 mg) in 50%yield as a semi-solid. NMR (CDCl₃, δ): 0.8 (dd, 3H), 1.2 (2T, 3H), 1.4(d, 3H), 1.7 (d, 3H), 1.8 (m, 2H), 2.1 (m, 1H), 3.5 (m, 6H), 4.0 (d,2H), 4.1-4.4 (m, 2H), 4.8 (m, 1H) and 5.4 (m, 2H).

EXAMPLE 10

Preparation of 2R,3R,5E-2-ol-3-methyl-5-hepten-1-glycyl carbamate

Compound 9 (γ=CH(CH₃)OC₂ H₅, p=1) 100mg was reacted with a hydrochloricacid (1N) in THF for one hour to give Compound 10 (p=1) as a whitecrystal (m.p. 101°-102° C.) in 95% yield. NMR (CDCl₃, δ): 0.8 (d, 3H),1.6 (d, 3H), 1.8 (m, 1H), 2.2 (m, 2H), 3.4 (m, 1H), 3.7 (d, 2H), 3.8-4.1(m, 2H) and 5.4 (m, 2H). IR (KBr): 3400 bm, 3000 bm, 1730 s, 1680 S,1540 s, 1460 w, 1450 w, 1410 m, 1220 s, 1000 m cm⁻¹.

EXAMPLE 11

Conjugation of Compound 10 (p=l) to G6PDH

Compound 10 (p=l) (24.5 mg) was reacted with N-hydroxy succinimide (11.5mg) in DMF overnight at 0°-5° C. and then was added portionwise to asolution of glucose-6-phosphate dehydrogenase (G6PDH) andglucose-6-phosphate (G6P) and NADH in a Tris base buffer at pH 8.0 andice bath temp. Two samples at 46% and 70% deactivation of enzyme wereprepared. The samples were purified on a Sephadex G-25 column using Trisbase buffer pH 8.0. The hapten numbers were 5 and 10 for 46% and 70%deactivation of enzyme.

EXAMPLE 12

Conjugation of Compound 10 (p=1) to Keyhole Limpet Hemocyanin (KLH)

To a stirred solution of radioactive labeled Compound 10 (p=1) (24.5 mg,0.1 mmol, 2.19×10⁷ CPm/mmol) in DMF (2 ml) was added N-hydroxysuccinimide (11.7 mg, 0.11 mmol). The mixture was then cooled in an icebath and dicyclohexyl carbodiimide (DCC) (20.6 mg, 0.11 mmol) was added.The mixture was stirred overnight in a cold room (+5° C.). The reactionmixture was then filtered and was added to a suspension of KLH (910) mgin sodium borate buffer (30 ml, pH=8.5). The mixture was stirred in thecold room overnight and then it was concentrated to half its volume(Amicon filter, PM 30,000). The mixture was purified on a Sephadex G-50column using water as eluting solvent. The protein fraction was thencollected and dialyzed in water and then was lyophilized to give 500 mgof the protein. The hapten/protein ratio was determined to be 170.

EXAMPLE 13

Preparation of Monoclonal Antibodies

The standard hybridoma procedures used have been described in detail byHurrell (Hurrell, J. G. R. "Monoclonal Hybridoma Antibodies: Techniquesand Applications" (1982) CRC Press, Boca Raton, Fla. 33431).

The cell lines used were P3-X63-AG8.653 myeloma cells and spleen cellsfrom 4-Balb/c mice immunized with the conjugate of Example 12, namely,Compound 10 (p=1)-KLH conjugate, as an immunogen. Mice received 3-100 μgdoses of the immunogen in Freund's adjuvant by intraperitoneal (IP)injection at monthly intervals, followed by a final IP booster in salinethree days before the fusion. Cells were fused using polyethylene glycoland then were suspended in HAT media and distributed into 20 96-wellmicroplates. Media in the wells were replenished four days later, andthe resultant hybridomas were screened for specific antibodies when thewells were half-confluent with cells, seven days after the fusion.

Cell lines selected for further study were rigorously cloned out atleast three times by a limiting dilution method before being expandedfor ascites production or scale-up by in vitro techniques.

Reverse and Competitive ELISA assays were used to establish thespecificity and usefulness of the monoclonal antibodies produced.

EXAMPLE 14

Assays

For the forward ELISA assays, Costar EIA plates were coated with 50μL/well affinity purified rabbit anti-mouse IgG+IgM+IgA antisera (1.0mg/ml stock diluted 1:100 in phosphate buffered saline (PBS)) andincubated at 37° C. for one hour. The plates were blocked by adding 300μL/well of 1% normal sheep sera (NSS) in PBS and incubated as before.The liquid was then shaken from the plate, and 50 μL/well of spent mediaantibody was then added and incubated as in the previous step. Theplates were then washed three times using the ELISA wash buffer (PBSplus 0.05% Tween 20, pH 7.2). In the reverse ELISA assay, 50 μL/well ofsynthetic C9 amino acidanalog-G6PDH conjugate (diluted 1:400 in PBS) wasadded. When screening by the Competitive ELISA assay, 50 μL/well of thesynthetic C9 amino acid analog of Example 10 in 100 μg/mL of dilutedenzyme conjugate was added. The plates were incubated for 30 minutes at37° C., and washed three times. 75 μL/well of substrate (0.1275M Tris,0.04M NAD, 0.066M G-6-P, 0.3 mg/mL INT [4-iodo 2,4-nitrophenyl3-5-phenoltetrazolium chloride], and 0.6 IU/mL diaphorase with 1% NSS)was then added and incubated until adequate color developed. Theabsorbance of each well at 492 nm was read.

The panel of monoclonal antibodies selected in the ELISA assays was nextexamined for performance in an EMIT® assay protocol. The EMIT assayprotocol is described in U.S. Pat. No. 3,517,837. Stock calibrators ofsynthetic C9 amino acid analog containing 0, 5, and 20 μg/mL werediluted 1:5 in assay buffer and 50 μL of the diluted calibrator andvarious amounts of spent media antibody were combined and diluted to 250uL with assay buffer. NAD and glucose-6-phosphate substrate and theenzyme conjugate were added, 50 μL each +250 μL assay buffer and thechange in absorbence at 340 nm was measured over a 30 second period. Theresults for antibody designated Cy XV 8C4 are summarized in (Table 1).

                  TABLE 1                                                         ______________________________________                                        Performance of Cy XV 8C4 Spent Media Antibody                                 in EMIT* Assay                                                                Antibody  Absorbence at 340 mm                                                (μL)   0 μg/ml   15 μg/ml                                                                            20 μg/ml                                   ______________________________________                                        250       227          244      472                                           150       358          367      590                                            50       633          651      744                                                     783                                                                 ______________________________________                                    

The invention provides for the preparation of reagents useful in anassay for the determination of cyclosporin, wherein the preparation ofthe reagents can be carried out in a relatively short number of steps.The reagents can be prepared in an optically active state andenantiomerically pure with good yields.

Although the foregoing invention has been described in some detail byway of illustration and example for the purpose of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A compound of the formula: ##STR10## wherein: Xis a linking group of 0-65 atoms not counting hydrogen, said atoms beingindependently selected from the group consisting of O, S, P, N, Si, Cand halogen,Z is a luminescent label, a group of molecular weightgreater than 5000 which is an immunogenic carrier or an enzyme, or aligand, or a hapten; Y is oxygen or sulfur; and n is a number from 1 upto the molecular weight of Z divided by 1000; and including theoptically active isomers thereof.
 2. The compound of claim 1 wherein Yis oxygen.
 3. The compound of claim 2 wherein X is --CH₂ OA and A is alinking group of 0-63 atoms not counting hydrogen and is bonded to --CH₂O through a carbon atom and is bonded to Z.
 4. The compound of claim 3wherein A contains a nonoxocarbonyl bonded to Z.
 5. The compound ofclaim 3 wherein said carbon atom is bonded to W as C=W and W is O, S orNR and R is H or lower alkyl.
 6. The compound of claim 5 wherein saidC=W is bonded to NRX' and X' is a linking group having from 0 to 60atoms not counting hydrogen.
 7. The compound of claim 1 wherein Zcomprises a poly(amino acid).
 8. The compound of claim 1 wherein Z is anenzyme.
 9. The compound of claim 1 wherein Z is glucose-6-phosphatedehydrogenase.
 10. The compound of claim 9 wherein n is a number from 4to
 10. 11. The compound of claim 1 wherein Z is an immunogenic carrierselected from the group consisting of immunogenic poly(amino acids),lipopolysaccharides, and particles which are 0.01 microns to 100 micronsin diameter.
 12. The compound of claim 1 wherein Z is a luminescentlabel selected from the group consisting of fluorogenic substrates,fluorescers, chemiluminescers, and fluorescent particles.
 13. A compoundof the formula: ##STR11## wherein: X₁ is a linking group of 0 to 35atoms not counting hydrogen, which atoms are each independently selectedfrom the group consisting of carbon, oxygen, nitrogen, and sulfur,wherein X₁ is bonded to O through a carbon atom;Z₁ is a poly(amino acid)of molecular weight greater than 5000 which is an antigen or an enzyme;n₁ is a number from 1 up to the molecular weight of Z₁ divided by 1000;including the optically active isomers thereof.
 14. The compound ofclaim 13 wherein X₁ includes non-oxocarbonyl group.
 15. The compound ofclaim 13 wherein X₁ comprises a carbamate functional group.
 16. Thecompound of claim 13 wherein X₁ is a linking group of from 2 to 10 atomsnot counting hydrogen.
 17. The compound of claim 13 wherein Z₁ is apoly(amino acid) which is an antigen.
 18. The compound of claim 13wherein Z_(l) is selected from the group consisting of an albumin and agamma globulin.
 19. The compound of claim 13 wherein Z₁ is an enzyme.20. The compound of claim 13 wherein Z₁ is glucose-6-phosphatedehydrogenase.
 21. The compound of claim 13 wherein n₁ is a number from4 to
 12. 22. The compound of claim 13 which has an isomericconfiguration of (2R, 3R).
 23. A compound of the formula: ##STR12##wherein: W₁ is O, S, or NH;m is 0 to 10; Z₂ is a poly(amino acid) ofmolecular weight greater than 5000 which is an antigen or an enzyme; n₂is a number from 1 up to the molecular weight of Z₂ divided by 1000;including the optical isomers thereof,
 24. The compound of claim 23wherein W₁ is
 0. 25. The compound of claim 23 wherein m is 1 to
 5. 26.The compound of claim 23 wherein n₂ is 4 to
 12. 27. The compound ofclaim 23 wherein Z₂ is glucose-6-phosphate dehydrogenase.
 28. Thecompound of claim 23 wherein Z₂ is an immunoglobulin.
 29. The compoundof claim 23 which has an isomeric configuration of (2R, 3R).